It is generally recognized that human milk is the best nutritional source for human infants. Human milk is not only an ideal source of nutrients for the developing infant, but also contains both immunoglobulins and non-immunological factors that protect the infant from infection by various organisms. Human milk is also easily digested by the infant and is less likely to cause allergic reactions than is infant formula based on bovine milk.
Human milk differs from bovine milk as well as the milk of other mammalian species in various ways. Overall protein content and the kinds of protein differ between human and bovine milk. Four major bovine caseins have been identified. Bovine milk contains 2 .alpha.-caseins plus .beta.- and .chi.-casein, but human milk contains only .beta.- and .chi.-casein. Additionally, the amino acid sequences of human milk protein differ from that of other mammalian milk proteins.
Efforts have been made to develop infant milk formulas that have some of the advantageous properties of human milk and avoid the disadvantages associated with bovine milk based infant formulas such as allergic reactions and incomplete digestion by the infant. An intuitively desirable method to achieve this is to add to the formula some of the known constituents of human milk, including human milk proteins in their native form. The human caseins, which differ in amino acid sequence from their bovine and other mammalian counterparts, represent important substances which, if added in their native form to infant formula, would serve to enhance the nutritional value of the formula and reduce the inherent disadvantages of non-human milk proteins.
In addition to being a source of amino acids necessary for the synthesis of proteins required for the growth and development of infants, human milk is recognized as containing proteins, including casein, that have other important biological functions. .beta.-casein is one of the most abundant milk proteins synthesized in the mammary gland. After post-translational modification in the Golgi apparatus, it is excreted as large calcium-dependent aggregates called micelles. .beta.-casein is not a single entity, but is a heterogeneous group of phosphoproteins secreted during lactation in response to lactogenic hormones. The primary structure of human .beta.-casein was determined by Greenberg et al.(Journal of Biological Chemistry 259:5132-5138, 1984). It was shown to be a phosphorylated protein with phosphorylation sites at specific seryl and threonyl residues located near the amino terminus. Comparison of human and bovine .beta.-caseins showed 47% identity. The sequence of human .chi.-casein was determined by Brignon et al. (Federation of European Biological Societies Letters 188:48-54, 1985). Whereas .beta.-casein is phosphorylated, .chi.-casein is glycosylated.
Several biological effects have been ascribed to human milk casein including: (1) enhancement of calcium absorption; (2) inhibition of angiotensin I-converting enzyme; (3) opioid agonism; (4) and immunostimulating and immunomodulating effects.
Human casein consists largely (&gt;80%) of the .beta.-form with a smaller amount in the .chi.-form (Greenberg et al., 1984). Native .beta.-casein is a 25 kDa protein. In human milk, .beta.-casein molecules show variable degrees of post-translational phosphorylation ranging from zero to five phosphate groups per polypeptide chain (Greenberg et al., 1984; Hansson et al., Protein Expression and Purification 4:373-381, 1993). Phosphate groups in the native protein are attached to serine and threonine residues located near the amino terminus (Greenberg et al., 1984).
Expression of exogenous genes in bacterial cells provides a useful method for producing recombinant eukaryotic proteins. However, bacteria, such as E. coli, are not capable of producing the post-translational modifications required by many eukaryotic proteins as they do not possess the endogenous enzymes necessary to do so. Therefore, eukaryotic proteins produced in E. coli lack the specific post-translational modifications which may occur within the eukaryotic cell, such as glycosylation, phosphorylation, acetylation, or amidation.
Prior to the development of appropriate cloning techniques, the phosphorylation of purified proteins by a kinase was done in vitro using chemical reagents. This process requires the protein substrate and the kinase enzyme to be purified and this is not efficient or cost-effective for commercial purposes. The in vitro process is also inefficient when it is desired to scale-up for commercialization. There is, therefore, a need to develop a method for genetically engineering microorganisms to phosphorylate a protein in vivo.
Canadian Patent Application No. 2,083,521 to Pawson et al. teaches a method of producing phosphorylated exogenous protein in host cells. The method of Pawson et al. requires two vectors to be introduced into a bacterial cell. One vector has a nucleotide sequence encoding an exogenous protein that is capable of being phosphorylated by the catalytic domain of a protein kinase. The other vector has a nucleotide sequence encoding the protein kinase catalytic domain. Both vectors are introduced into E. coli and production of the exogenous protein and the protein kinase catalytic domain is induced so that the exogenous protein is phosphorylated. The bacterial cells are then lysed and the exogenous phosphorylated protein is isolated using standard isolation techniques.
CA No. 2,083,521 does not suggest or disclose the method of the instant invention. The present inventors use a single vector expressing both the substrate and the kinase enzyme. The method of Pawson et al. requires the use of two vectors. The expression system disclosed herein results in specific phosphorylation of the exogenous protein as determined by antibody to phosphoserine, while the expression system of Pawson et al. results in non-specific phosphorylation of both host proteins and exogenous proteins. This would adversely affect the growth of host bacteria in scale-up efforts for industrial applications. The present invention, unlike that of Pawson et al., provides for high level production of a phosphorylated, recombinant protein suitable for commercial production.
Simcox et al., Strategies in molecular biology 7(3):68-69 (1994) constructed two E. coli strains that harbor a tyrosine kinase plasmid. These TK (tyrosine kinase) strains can be used for generating phosphorylated proteins when transformed with a plasmid containing sequences encoding a phosphorylation target domain or protein. Both E. coli strains carry an inducible tyrosine kinase gene. One strain, TKB1, is useful for expressing genes whose expression is directed by the T7 promoter. The system developed by Simcox et al. differs from the present invention in that it requires two constructs, i.e., a tyrosine kinase-containing plasmid and a plasmid vector containing a gene encoding a protein or domain to be phosphorylated.
In order to better understand the structure and function of human .beta.-casein and to permit studies of factors that affect regulation of its synthesis and secretion, cDNA for this protein was cloned and sequenced (Lonnerdal et al., Federation of European Biological Societies Letters 269:153-156,1990), and human milk .beta.-casein was produced in Escherichia coli and Saccharomyces cerevisiae (Hansson et al., 1993). Hansson et al. demonstrated that recombinant human .beta.-casein was expressed in the yeast, S. cerevisiae, using the pYES 2.0 vector (Invitrogen Corp., San Diego, Calif.). Production levels were estimated to be approximately 10% of the production found in E. coli. However, recombinant .beta.-casein obtained from S. cerevisiae, a eukaryotic cell that has endogenous enzymes capable of phosphorylating proteins, was phosphorylated, but the protein produced by E. coli, a prokaryotic cell that lacks the ability in its native state to phosphorylate, was non-phosphorylated. Subsequently, it was shown that recombinant human casein kinase II (rhCKII) produced in and purified from E. coli can phosphorylate protein substrates in vitro (Shi et al., Proceeding of the National Academy of Sciences, USA 91:2767-2771, 1994). One specific embodiment of the present invention uses a nucleotide sequence encoding a recombinant human casein kinase II in a single construct with nucleotide sequence encoding .beta.-casein to transform E. Coli and produce phosphorylated .beta.-casein. None of the prior art suggests or discloses a single vector containing a promoter followed by a nucleotide sequence encoding a protein followed by a nucleotide sequence encoding a kinase as is disclosed in the present invention.